Biomarkers and diagnostic methods for alzheimer&#39;s disease and other neurodegenerative disorders

ABSTRACT

The present invention relates to biomarkers and diagnostic and prognostic methods for Alzheimer&#39;s disease and other neurodegenerative disorders. The invention also provides compositions for detecting the biomarker as well as compositions and methods useful for treating Alzheimer&#39;s disease and other neurodegenerative disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/522,585, filed on Oct. 24, 2014, which claims priority to the U.S.Provisional Patent Application Ser. No. 61/895,376, filed on Oct. 24,2013, U.S. Provisional Patent Application Ser. No. 61/978,994, filed onApr. 14, 2014, and U.S. Provisional Patent Application Ser. No.62/047,062, filed on Sep. 8, 2014. These applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to biomarkers and diagnostic andprognostic methods for Alzheimer's disease and other neurodegenerativedisorders. The invention also provides compositions for detecting thebiomarker as well as compositions and methods useful for treatingAlzheimer's disease and other neurodegenerative disorders.

BACKGROUND OF THE INVENTION

More than 5.4 million Americans and 35 million people worldwide haveAlzheimer's disease, the most common form of dementia. Currently, theonly definitive way to diagnose Alzheimer's disease is by directexamination of brain tissue after a patient dies. Doctors use brainimaging, evaluation of behavior, psychiatric tests, and other means todiagnose the disease in the patients suspected of having Alzheimer'sdisease, but none are highly accurate, and many are costly or notpractical.

Therefore, there is a need in the art for biomarkers and methods fordiagnosing Alzheimer's disease and other neurodegenerative disorders.Additionally, there is a need in the art for compositions for detectingthe biomarkers as well as compositions and methods useful for treatingAlzheimer's disease and other neurodegenerative disorders. The presentinvention meets this need by providing accurate, noninvasive methods fordiagnosing Alzheimer's disease and other neurodegenerative disorders.The present invention further provides novel methods, assays, kits, andcompositions for diagnosing, prognosing, predicting, and treatingAlzheimer's disease and other neurodegenerative disorders.

SUMMARY OF THE INVENTION

The present invention provides methods of diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder, comprising: assaying the level of one or more biomarkers in abiological sample from the subject; and diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder based on the levels of the biomarker, wherein at least one ofthe one or more biomarkers are selected from the group consisting ofTau, phosphorylated Tau, Aß1-42, TDP-43, α-synuclein, SOD-1, FUS,FKBP51, IRS-1, phosphorylated IRS-1, cathepsin D (CTSD), type 1lysosome-associated membrane protein (LAMP1), ubiquitinylated proteins(UBP), heat-shock protein 70 (HSP70), neuron-specific enolase (NSE),neurofilament light chain (NFL), CD9, CD63, CD81, and CD171. In someembodiments, the level of the one or more biomarkers in the biologicalsample is compared to the level of one or more biomarkers in a controlsample and wherein the level of the one or more biomarkers of thebiological sample is elevated compared to the control sample. In otherembodiments, the neurodegenerative disorder is selected from the groupconsisting of: Alzheimer's disease (AD), vascular disease dementia,frontotemporal dementia (FTD), corticobasal degeneration (CBD),progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease. In other embodiments, the biological sample isselected from the group consisting of whole blood, serum, plasma, urine,interstitial fluid, peritoneal fluid, cervical swab, tears, saliva,buccal swab, skin, brain tissue, and cerebrospinal fluid. In yet otherembodiments, the phosphorylated Tau is phosphorylated on one or moreserine, threonine, or tyrosine residues. In still other embodiments, thephosphorylated Tau is phosphorylated at one or more residues selectedfrom the group consisting of Thr-153, Thr-181, Thr-205, Thr-231,Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, Ser-422,Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In other embodiments, thelevel of phosphorylated Tau is determined by assaying the level ofphosphorylated Tau polynucleotide, phosphorylated Tau polypeptide, orphosphorylated Tau activity. In yet other embodiments, thephosphorylated IRS-1 is phosphorylated on one or more serine, threonine,or tyrosine residues. In still other embodiments, the phosphorylatedIRS-1 is phosphorylated at one or more residues selected from the groupconsisting of P-S312-IRS-1 and P-panY-IRS-1. In other embodiments, thelevel of phosphorylated IRS-1 is determined by assaying the level ofphosphorylated IRS-1 polynucleotide, phosphorylated IRS-1 polypeptide,or phosphorylated IRS-1 activity. In other embodiments, the methodfurther comprises isolating vesicles from the biological samples. Inother embodiments, the vesicles are selected from the group consistingof exosomes, microparticles, microvesicles, nanosomes, extracellularvesicles, and ectosomes. In other embodiments, the method furthercomprises isolating exosomes from the biological sample. In certainembodiments, the isolated exosomes are selected from the groupconsisting of neuron-derived exosomes, astrocyte-derived exosomes,oliogodendrocyte-derived exosomes, and microglia-derived exosomes. Inother embodiments, the level of one or more biomarkers is the protein,phosphorylated protein, mRNA, or miRNA level of the one or morebiomarker.

The present invention also provides methods of diagnosing or prognosinga neurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder, comprising: isolating vesicles from a biological sampleobtained from the subject; and determining the level of one or morebiomarkers in the vesicles; wherein an elevated level of the one or morebiomarkers in the sample compared to the level of the one or morebiomarkers in a control sample is an indication of a neurodegenerativedisorder, wherein at least one of the one or more biomarkers is selectedfrom the group consisting of Tau, phosphorylated Tau, Aß1-42, TDP-43,α-synuclein, SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1, CTSD,LAMP1, UBP, HSP70, NSE, NFL, CD9, CD63, CD81, and CD171. In otherembodiments, the vesicles are selected from the group consisting ofexosomes, microparticles, microvesicles, nanosomes, extracellularvesicles, and ectosomes. In some embodiments, the neurodegenerativedisorder is selected from the group consisting of: Alzheimer's disease(AD), vascular disease dementia, frontotemporal dementia (FTD),corticobasal degeneration (CBD), progressive supranuclear palsy (PSP),Lewy body dementia, tangle-predominant senile dementia, Pick's disease(PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS),other motor neuron diseases, Guam parkinsonism-dementia complex,FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic braininjury (TBI), and Parkinson's disease. In other embodiments, thebiological sample is selected from the group consisting of whole blood,serum, plasma, urine, interstitial fluid, peritoneal fluid, cervicalswab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinalfluid. In yet other embodiments, the phosphorylated tau isphosphorylated on one or more serine, threonine, or tyrosine residues.In other embodiments, the phosphorylated Tau is phosphorylated at one ormore residues selected from the group consisting of Thr-153, Thr-181,Thr-205, Thr-231, Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356,Ser-396, Ser-422, Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In yetother embodiments, the phosphorylated IRS-1 is phosphorylated on one ormore serine, threonine, or tyrosine residues. In other embodiments, thephosphorylated IRS-1 is phosphorylated at one or more residues selectedfrom the group consisting of P-S312-IRS-1 and P-panY-IRS-1. In stillother embodiments, the isolated exosomes are selected from the groupconsisting of neuron-derived exosomes, astrocyte-derived exosomes,oliogodendrocyte-derived exosomes, and microglia-derived exosomes. Inother embodiments, the isolating vesicles from a biological samplecomprises: contacting the biological sample with an agent underconditions wherein a vesicle present in said biological sample binds tosaid agent to form a vesicle-agent complex; and isolating said vesiclefrom said vesicle-agent complex to obtain a sample containing saidvesicle, wherein the purity of vesicles present in said sample isgreater than the purity of vesicles present in said biological sample.In other embodiments, the isolating vesicles from a biological samplecomprises: isolating vesicles from said biological sample to obtain avesicle sample; contacting the vesicle sample with an agent underconditions wherein a vesicle present in said vesicle sample binds tosaid agent to form a vesicle-agent complex; and isolating said vesiclefrom said vesicle-agent complex to obtain a sample containing saidvesicle, wherein the purity of vesicles present in said sample isgreater than the purity of vesicles present in said biological sample.In certain aspects, the agent is an antibody, a lectin, a ligand, asoluble receptor, a binding protein, or an oligonucleotide. In otheraspects, the antibody is a polyclonal or monoclonal antibody. In yetother aspects, the antibody is a monoclonal NCAM antibody. In otheraspects, the antibody is a monoclonal anti-human NCAM antibody. In yetother aspects, the antibody is a monoclonal CD171 antibody. In otheraspects, the antibody is a monoclonal anti-human CD171 antibody. Inother aspects, the antibody is a monoclonal CD9 antibody. In otheraspects, the antibody is a monoclonal CD63 antibody. In other aspects,the antibody is a monoclonal CD81 antibody. In other embodiments, thevesicles are selected from the group consisting of exosomes,microparticles, microvesicles, nanosomes, extracellular vesicles, andectosomes. In other embodiments, the level of one or more biomarkers isthe protein, phosphorylated protein, mRNA, or miRNA level of the one ormore biomarker.

The present invention provides methods of diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder, comprising: obtaining a biological sample from the subject;applying an antibody specific for vesicles to the sample, wherein thepresence of the vesicle creates an antibody-vesicle complex; isolatingthe antibody-vesicle complex; assaying a level of one or more biomarkersin the antibody-vesicle complex; and diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder based on the levels of the one or more biomarkers, wherein atleast one of the biomarkers are selected from the group consisting ofTau, phosphorylated Tau, Aß1-42, TDP-43, α-synuclein, SOD-1, FUS,FKBP51, IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP, HSP70, NSE, NFL,CD9, CD63, CD81, and CD171. In some embodiments, the antibody-vesiclecomplex is created on a solid phase. In yet other embodiments, themethods further comprise releasing the vesicle from the antibody-vesiclecomplex. In certain embodiments, the solid phase is non-magnetic beads,magnetic beads, agarose, or sepharose. In other embodiments, the vesicleis released by exposing the antibody-vesicle complex to low pH between3.5 and 1.5. In yet other embodiments, the released vesicle isneutralized by adding a high pH solution. In other embodiments, thereleased vesicle is lysed by incubating the released vesicles with alysis solution. In still other embodiments, the lysis solution containsinhibitors for proteases and phosphatases. In other embodiments, thelevels of the one or more biomarkers are normalized by the number ofvesicles or values of vesicle biomarkers. In other embodiments, thevesicles are selected from the group consisting of exosomes,microparticles, microvesicles, nanosomes, extracellular vesicles, andectosomes. In certain embodiments, the antibody is a polyclonal ormonoclonal antibody. In other embodiments, the antibody is a monoclonalNCAM antibody. In other embodiments, the antibody is a monoclonalanti-human NCAM antibody. In yet other aspects, the antibody is amonoclonal CD171 antibody. In other aspects, the antibody is amonoclonal anti-human CD171 antibody. In other aspects, the antibody isa monoclonal CD9 antibody. In other aspects, the antibody is amonoclonal CD63 antibody. In other aspects, the antibody is a monoclonalCD81 antibody. In some embodiments, the exosomes are selected from thegroup consisting of neuron-derived exosomes, astrocyte-derived exosomes,oliogodendrocyte-derived exosomes, and microglia-derived exosomes. Inother embodiments, the phosphorylated tau is phosphorylated on one ormore serine, threonine, or tyrosine residues. In still otherembodiments, the phosphorylated Tau is phosphorylated at one or moreresidues selected from the group consisting of Thr-153, Thr-181,Thr-205, Thr-231, Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356,Ser-396, Ser-422, Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In otherembodiments, the phosphorylated IRS-1 is phosphorylated on one or moreserine, threonine, or tyrosine residues. In still other embodiments, thephosphorylated IRS-1 is phosphorylated at one or more residues selectedfrom the group consisting of P-S312-IRS-1 and P-panY-IRS-1. In yet otherembodiments, the biological sample is selected from the group consistingof whole blood, serum, plasma, urine, interstitial fluid, peritonealfluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue,and cerebrospinal fluid. In some embodiments, the neurodegenerativedisorder is selected from the group consisting of: Alzheimer's disease(AD), vascular disease dementia, frontotemporal dementia (FTD),corticobasal degeneration (CBD), progressive supranuclear palsy (PSP),Lewy body dementia, tangle-predominant senile dementia, Pick's disease(PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS),other motor neuron diseases, Guam parkinsonism-dementia complex,FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic braininjury (TBI), and Parkinson's disease. In other embodiments, the levelof one or more biomarkers is the protein, phosphorylated protein, mRNA,or miRNA level of the one or more biomarker.

The present invention provides sets of biomarkers for assessingneurodegenerative disorder status of a subject comprising one or morebiomarkers, wherein the levels of the biomarkers in the set are assayed;and wherein the biomarker level determines the neurodegenerativedisorder status of the subject with at least 40% specificity, whereinthe at least one or more of the set of biomarkers are selected from thegroup consisting of Tau, phosphorylated Tau, Aß1-42, TDP-43,α-synuclein, SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1, CTSD,LAMP1, UBP, HSP70, NSE, NFL, CD9, CD63, CD81, and CD171. In someembodiments, the biomarker level determines the neurodegenerativedisorder status of the subject with at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 99%, or 100% specificity. the In some embodiments,the biological sample is selected from the group consisting of wholeblood, serum, plasma, urine, interstitial fluid, peritoneal fluid,cervical swab, tears, saliva, buccal swab, skin, cerebrospinal fluid. Inother embodiments, the neurodegenerative disorder is selected from thegroup consisting of: Alzheimer's disease (AD), vascular diseasedementia, frontotemporal dementia (FTD), corticobasal degeneration(CBD), progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease. In yet other embodiments, the methods furthercomprise assaying the levels of the biomarkers in vesicles from thesample. In other embodiments, the vesicles are selected from the groupconsisting of exosomes, microparticles, microvesicles, nanosomes,extracellular vesicles, and ectosomes.

The present invention also provides kits for diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder, the kit comprising one or more agents which specifically bindsvesicles, one or more agents which specifically bind a biomarker, one ormore containers for collecting and or holding the biological sample, andan instruction for its use, wherein the neurodegenerative disorder isassociated with altered biomarker levels and wherein the biomarker isselected from the group consisting of Tau, phosphorylated Tau, Aß1-42,TDP-43, α-synuclein, SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1,CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9, CD63, CD81, and CD171. In otherembodiments, the vesicles are selected from the group consisting ofexosomes, microparticles, microvesicles, nanosomes, extracellularvesicles, and ectosomes. In some embodiments, the agents are polyclonalor monoclonal antibodies. In other embodiments, the antibodies are amonoclonal NCAM antibody. In other embodiments, the antibody is amonoclonal anti-human NCAM antibody. In yet other aspects, the antibodyis a monoclonal CD171 antibody. In other aspects, the antibody is amonoclonal anti-human CD171 antibody. In other aspects, the antibody isa monoclonal CD9 antibody. In other aspects, the antibody is amonoclonal CD63 antibody. In other aspects, the antibody is a monoclonalCD81 antibody. In certain embodiments, the exosomes are selected fromthe group consisting of neuron-derived exosomes, astrocyte-derivedexosomes, oliogodendrocyte-derived exosomes, and microglia-derivedexosomes. In other embodiments, the neurodegenerative disorder isselected from the group consisting of: Alzheimer's disease (AD),vascular disease dementia, frontotemporal dementia (FTD), corticobasaldegeneration (CBD), progressive supranuclear palsy (PSP), Lewy bodydementia, tangle-predominant senile dementia, Pick's disease (PiD),argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), othermotor neuron diseases, Guam parkinsonism-dementia complex, FTDP-17,Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI),and Parkinson's disease. In yet other embodiments, the biological sampleis selected from the group consisting of whole blood, serum, plasma,urine, interstitial fluid, peritoneal fluid, cervical swab, tears,saliva, buccal swab, skin, cerebrospinal fluid. In still otherembodiments, the phosphorylated tau is phosphorylated on one or moreserine, threonine, or tyrosine residues. In other embodiments, thephosphorylated Tau is phosphorylated at one or more residues selectedfrom the group consisting of Thr-153, Thr-181, Thr-205, Thr-231,Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, Ser-422,Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In still other embodiments,the phosphorylated IRS-1 is phosphorylated on one or more serine,threonine, or tyrosine residues. In other embodiments, thephosphorylated IRS-1 is phosphorylated at one or more residues selectedfrom the group consisting of P-S312-IRS-1 and P-panY-IRS-1. In otherembodiments, the kits further comprise a computer model or algorithm foranalyzing the biomarker level in the sample.

The present invention also provides kits for diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder, the kit comprising one or more agents which specifically bindsvesicles, one or more probes or primers for detecting biomarker mRNA ormiRNA, one or more containers for collecting and or holding thebiological sample, and an instruction for its use, wherein theneurodegenerative disorder is associated with altered biomarker levelsand wherein the biomarker is selected from the group consisting of Tau,phosphorylated Tau, Aß1-42, TDP-43, α-synuclein, SOD-1, FUS, FKBP51,IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9,CD63, CD81, and CD171. In other embodiments, the vesicles are selectedfrom the group consisting of exosomes, microparticles, microvesicles,nanosomes, extracellular vesicles, and ectosomes. In some embodiments,the agents are polyclonal or monoclonal antibodies. In otherembodiments, the exosomes are selected from the group consisting ofneuron-derived exosomes, astrocyte-derived exosomes,oliogodendrocyte-derived exosomes, and microglia-derived exosomes. Inyet other embodiments, the neurodegenerative disorder is selected fromthe group consisting of: Alzheimer's disease (AD), vascular diseasedementia, frontotemporal dementia (FTD), corticobasal degeneration(CBD), progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease. In still other embodiments, the biological sampleis selected from the group consisting of whole blood, serum, plasma,urine, interstitial fluid, peritoneal fluid, cervical swab, tears,saliva, buccal swab, skin, cerebrospinal fluid. In other embodiments,the phosphorylated tau is phosphorylated on one or more serine,threonine, or tyrosine residues. In still other embodiments, thephosphorylated Tau is phosphorylated at one or more residues selectedfrom the group consisting of Thr-153, Thr-181, Thr-205, Thr-231,Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, Ser-422,Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In other embodiments, thephosphorylated IRS-1 is phosphorylated on one or more serine, threonine,or tyrosine residues. In still other embodiments, the phosphorylatedIRS-1 is phosphorylated at one or more residues selected from the groupconsisting of P-S312-IRS-1 and P-panY-IRS-1. In other embodiments, thekits further comprise a computer model or algorithm for analyzing thebiomarker level in the sample.

In other embodiments, the present invention provides methods ofdiagnosing a neurodegenerative disorder in a subject, comprising thesteps of: (i) obtaining a test biological sample containing vesiclesfrom the subject, (ii) measuring the level of one or more biomarkers inthe test biological sample, (iii) comparing the level of the one or morebiomarkers in the test biological sample to a control level of the oneor more biomarkers in a control biological sample, and (iv) determiningthe subject has a neurodegenerative disorder by detecting an increasedlevel of the one or more biomarkers in the test biological sample,relative to the control biological sample, wherein at least one of theone or more biomarkers is selected from the group consisting of Tau,phosphorylated Tau, Aß1-42, TDP-43, α-synuclein, SOD-1, FUS, FKBP51,IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9,CD63, CD81, and CD171. In other embodiments, the vesicles are selectedfrom the group consisting of exosomes, microparticles, microvesicles,nanosomes, extracellular vesicles, and ectosomes. In some embodiments,the exosomes are selected from the group consisting of neuron-derivedexosomes, astrocyte-derived exosomes, oliogodendrocyte-derived exosomes,and microglia-derived exosomes. In other embodiments, the phosphorylatedtau is phosphorylated on one or more serine, threonine, or tyrosineresidues. In still other embodiments, the phosphorylated Tau isphosphorylated at one or more residues selected from the groupconsisting of Thr-153, Thr-181, Thr-205, Thr-231, Ser-199, Ser-202,Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, Ser-422, Tyr18, Tyr29,Tyr197, Tyr310, and Tyr 394. In other embodiments, the phosphorylatedIRS-1 is phosphorylated on one or more serine, threonine, or tyrosineresidues. In still other embodiments, the phosphorylated IRS-1 isphosphorylated at one or more residues selected from the groupconsisting of P-S312-IRS-1 and P-panY-IRS-1. In yet other embodiments,the neurodegenerative disorder is selected from the group consisting of:Alzheimer's disease (AD), vascular disease dementia, frontotemporaldementia (FTD), corticobasal degeneration (CBD), progressivesupranuclear palsy (PSP), Lewy body dementia, tangle-predominant seniledementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophiclateral sclerosis (ALS), other motor neuron diseases, Guamparkinsonism-dementia complex, FTDP-17, Lytico-Bodig disease, multiplesclerosis, traumatic brain injury (TBI), and Parkinson's disease. Instill other embodiments, the biological sample is selected from thegroup consisting of whole blood, serum, plasma, urine, interstitialfluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab,skin, cerebrospinal fluid. In other embodiments, the phosphorylated tauis phosphorylated on one or more serine, threonine, or tyrosineresidues. In other embodiments, the phosphorylated IRS-1 isphosphorylated on one or more serine, threonine, or tyrosine residues.In other embodiments, the method further comprises isolating vesiclesfrom the biological sample. In other embodiments, the vesicles areselected from the group consisting of exosomes, microparticles,microvesicles, nanosomes, extracellular vesicles, and ectosomes.

In other embodiments, the present invention provides methods foranalyzing a sample from a subject comprising the steps of: (i) obtaininga biological sample comprising vesicles from the subject, (ii) measuringthe level of one or more biomarkers in the biological sample, and (iii)comparing the level of the one or more biomarkers in the biologicalsample to a control level of the one or more biomarkers in a controlbiological sample. In some embodiments, the subject has been diagnosedor suspected of having a neurodegenerative disorder. In otherembodiments, the method further comprises diagnosing or prognosing aneurodegenerative disorder in the subject, identifying risk of aneurodegenerative disorder in the subject, or prescribing a therapeuticregimen or predicting benefit from therapy for the subject having orsuspected of having a neurodegenerative disorder. In certainembodiments, at least one of the one or more biomarkers are selectedfrom the group consisting of Tau, phosphorylated Tau, Aß1-42, TDP-43,α-synuclein, SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1, CTSD,LAMP1, UBP, HSP70, NSE, NFL, CD9, CD63, CD81, and CD171. In someembodiments, the level of the one or more biomarkers in the biologicalsample is compared to the level of one or more biomarkers in a controlsample and wherein the level of the one or more biomarkers of thebiological sample is elevated compared to the control sample. In otherembodiments, the neurodegenerative disorder is selected from the groupconsisting of: Alzheimer's disease (AD), vascular disease dementia,frontotemporal dementia (FTD), corticobasal degeneration (CBD),progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease. In other embodiments, the biological sample isselected from the group consisting of whole blood, serum, plasma, urine,interstitial fluid, peritoneal fluid, cervical swab, tears, saliva,buccal swab, skin, brain tissue, and cerebrospinal fluid. In yet otherembodiments, the phosphorylated Tau is phosphorylated on one or moreserine, threonine, or tyrosine residues. In still other embodiments, thephosphorylated Tau is phosphorylated at one or more residues selectedfrom the group consisting of Thr-153, Thr-181, Thr-205, Thr-231,Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, Ser-422,Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In other embodiments, thelevel of phosphorylated Tau is determined by assaying the level ofphosphorylated Tau polynucleotide, phosphorylated Tau polypeptide, orphosphorylated Tau activity. In yet other embodiments, thephosphorylated IRS-1 is phosphorylated on one or more serine, threonine,or tyrosine residues. In still other embodiments, the phosphorylatedIRS-1 is phosphorylated at one or more residues selected from the groupconsisting of P-S312-IRS-1 and P-panY-IRS-1. In other embodiments, thelevel of phosphorylated IRS-1 is determined by assaying the level ofphosphorylated IRS-1 polynucleotide, phosphorylated IRS-1 polypeptide,or phosphorylated IRS-1 activity. In other embodiments, the methodfurther comprises isolating vesicles from the biological samples. Inother embodiments, the vesicles are selected from the group consistingof exosomes, microparticles, microvesicles, nanosomes, extracellularvesicles, and ectosomes. In other embodiments, the method furthercomprises isolating exosomes from the biological sample. In certainembodiments, the isolated exosomes are selected from the groupconsisting of neuron-derived exosomes, astrocyte-derived exosomes,oliogodendrocyte-derived exosomes, and microglia-derived exosomes. Inother embodiments, the method further comprises determining a ratio ofP-S312-IRS-1 and P-panY-IRS-1 (R or insulin resistance index). In otherembodiments, the methods of the present invention further comprise acomputer model or algorithm for analyzing the one or more biomarkerlevel in the sample. In other embodiments, the level of one or morebiomarkers is the protein, phosphorylated protein, mRNA, or miRNA levelof the one or more biomarker.

These and other embodiments of the present invention will readily occurto those of skill in the art in light of the disclosure herein, and allsuch embodiments are specifically contemplated.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description. The invention is capable of other embodimentsand of being practiced or of being carried out in various ways. Also,the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing”, “involving”, andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items. It mustbe noted that as used herein and in the appended claims, the singularforms “a,” “an,” and “the” include plural references unless contextclearly dictates otherwise.

DESCRIPTION OF THE INVENTION

It is to be understood that the invention is not limited to theparticular methodologies, protocols, cell lines, assays, and reagentsdescribed herein, as these may vary. It is also to be understood thatthe terminology used herein is intended to describe particularembodiments of the present invention, and is in no way intended to limitthe scope of the present invention as set forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unlesscontext clearly dictates otherwise. Thus, for example, a reference to “afragment” includes a plurality of such fragments, a reference to an“antibody” is a reference to one or more antibodies and to equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Gennaro, A. R., ed. (1990) Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989)Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, ColdSpring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) ShortProtocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream etal., eds. (1998) Molecular Biology Techniques: An Intensive LaboratoryCourse, Academic Press); PCR (Introduction to Biotechniques Series), 2nded. (Newton & Graham eds., 1997, Springer Verlag).

The present invention relates, in part, to the discovery that exosomalbiomarkers can be assayed to identify subjects having or likely todevelop neurodegenerative disorders, including, for example, Alzheimer'sdisease (AD), multiple sclerosis (MS), and frontotemporal dementia(FTD).

The present invention is based, in part, on the discovery of unexpectedincreases in certain biomarkers in neuron-derived exosomes present inthe circulation of subjects having neurodegenerative disease (e.g.,Alzheimer's disease). The present invention demonstrates that exosomallevels of these biomarkers may be assayed to diagnose aneurodegenerative disorder in a subject having a neurodegenerativedisease. The present invention further shows that measurement of certainbiomarkers in neuron-derived exosomes from a subject may be used topredict the subsequent development of a neurodegenerative disease (e.g.,identify a subject at risk of developing a neurodegenerative disorder).

The present invention provides a set of biomarkers for assessingneurodegenerative status of a subject. In this embodiment, biomarkerlevels are assayed and the biomarker level determines theneurodegenerative status of the subject with at least 40% specificity.

The present invention also provides compositions for use in the methodsdescribed herein. Such compositions may include small moleculecompounds; peptides and proteins including antibodies or functionallyactive fragments thereof; and polynucleotides including smallinterfering ribonucleic acids (siRNAs), micro-RNAs (miRNAs), ribozymes,and anti-sense sequences. (See, e.g., Zeng (2003) Proc Natl Acad Sci USA100:9779-9784; and Kurreck (2003) Eur J Biochem 270:1628-1644.)

The present invention further provides kits for diagnosing or prognosinga neurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder. In these embodiments, the kits comprise one or more antibodieswhich specifically binds exosomes, one or more antibodies whichspecifically bind a biomarker, one or more containers for collecting andor holding the biological sample, and an instruction for the kits use.

The section headings are used herein for organizational purposes only,and are not to be construed as in any way limiting the subject matterdescribed herein.

Biological Sample

The present invention provides biomarkers and diagnostic and prognosticmethods for Alzheimer's disease and other neurodegenerative disorders.Biomarkers levels are determined in a biological sample obtained from asubject. In some embodiments, the biological sample of the invention canbe obtained from blood. In some embodiments, about 1-10 mL of blood isdrawn from a subject. In other embodiments, about 10 -50 mL of blood isdrawn from a subject. Blood can be drawn from any suitable area of thebody, including an arm, a leg, or blood accessible through a centralvenous catheter. In some embodiments, blood is collected following atreatment or activity. For example, blood can be collected following amedical exam. The timing of collection can also be coordinated toincrease the number and/or composition of exosomes present in thesample. For example, blood can be collected following exercise or atreatment that induces vascular dilation.

Blood may be combined with various components following collection topreserve or prepare samples for subsequent techniques. For example, insome embodiments, blood is treated with an anticoagulant, a cellfixative, a protease inhibitor, a phosphatase inhibitor, a protein, aDNA, or an RNA preservative following collection. In some embodiments,blood is collected via venipuncture using vacuum collection tubescontaining an anticoagulant such as EDTA or heparin. Blood can also becollected using a heparin-coated syringe and hypodermic needle. Bloodcan also be combined with components that will be useful for cellculture. For example, in some embodiments, blood is combined with cellculture media or supplemented cell culture media (e.g., cytokines).

Biological samples can also be obtained from other sources known in theart, including whole blood, serum, plasma, urine, interstitial fluid,peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin,cerebrospinal fluid, or other tissues including, for example, braintissues.

Enrichment or Isolation of Vesicles (Exosomes, Microparticles,Microvesicles, Nanosomes, Extracellular Vesicles, and Ectosomes)

Samples can be enriched for vesicles through positive selection,negative selection, or a combination of positive and negative selection.In some embodiments, vesicles are directly captured. In otherembodiments, blood cells are captured and vesicles are collected fromthe remaining biological samples. In some embodiments, the vesiclesenriched in the biological samples are exosomes, microparticles,microvesicles, nanosomes, extracellular vesicles, or ectosomes. In someembodiments, the vesicles enriched in the biological samples areneuron-derived exosomes, astrocyte-derived exosomes,oliogodendrocyte-derived exosomes, and microglia-derived exosomes.

Samples can also be enriched for vesicles based on differences in thebiochemical properties of vesicles. For example, samples can be enrichedfor vesicles based on antigen, nucleic acid, metabolic, gene expression,or epigenetic differences. In some of the embodiments based on antigendifferences, antibody-conjugated magnetic or paramagnetic beads inmagnetic field gradients or fluorescently labeled antibodies with flowcytometry are used. In some of the embodiments based on nucleic aciddifferences, flow cytometry is used. In some of the embodiments based onmetabolic differences, dye uptake/exclusion measured by flow cytometryor another sorting technology is used. In some of the embodiments basedon gene expression, cell culture with cytokines is used. Samples canalso be enriched for vesicles based on other biochemical propertiesknown in the art. For example, samples can be enriched for vesiclesbased on pH or motility. Further, in some embodiments, more than onemethod is used to enrich for vesicles. In other embodiments, samples areenriched for vesicles using antibodies, ligands, or soluble receptors.

In other embodiments, surface markers are used to positively enrichvesicles in the sample. In some embodiments, the vesicles are exosomes,microparticles, microvesicles, nanosomes, extracellular vesicles, orectosomes. In other embodiments, NCAM, CD171, CD9, CD63, CD81, diverseneuron or astrocyte adhesive proteins, microglial CD18/11, or CD3 T cellmembrane cell surface markers are used to enrich for exosomes. In someembodiments, cell surface markers that are not found on vesiclespopulations are used to negatively enrich vesicles by depleting cellpopulations. Flow cytometry sorting may also be used to further enrichfor exosomes using cell surface markers or intracellular orextracellular markers conjugated to fluorescent labels.

Intracellular and extracellular markers may include nuclear stains orantibodies against intracellular or extracellular proteinspreferentially expressed in vesicles. Cell surface markers may includeantibodies against cell surface antigens that are preferentiallyexpressed on exosomes (e.g., NCAM). In some embodiments, the cellsurface marker is a neuron-derived exosome surface marker, including,for example, NCAM or CD171. In some embodiments, a monoclonal NCAM, CD9,CD63, CD81 or CD171 antibody is used to enrich or isolate exosomes fromthe sample. In certain aspects, the NCAM, CD9, CD63, CD81 or CD171antibody is biotinylated. In this embodiment, biotinylated NCAM or CD171antibody can form an antibody-exosome complex that can be subsequentlyisolated using streptavidin-agarose resin or beads. In otherembodiments, the NCAM, CD9, CD63, CD81 or CD171 antibody is a monoclonalanti-human NCAM, CD9, CD63, CD81 or CD171 antibody.

In some embodiments, enriched vesicles from the biological sample aresubsequently enriched for a specific type of vesicle. For example, thebiological sample is enriched for exosomes and then the enrichedexosomes are subsequently enriched for neural-derived exosomes. In someembodiments, the biological sample is enriched for individual neuralcell sources of vesicles. In certain aspects, the neural cell sources ofvesicles are microglia, neurons, or astrocytes.

In other embodiments, vesicles are isolated or enriched from abiological sample comprising: contacting a biological sample with anagent under conditions wherein a vesicle present in said biologicalsample binds to said agent to form a vesicle-agent complex; andisolating said vesicle from said vesicle-agent complex to obtain asample containing said vesicle, wherein the purity of vesicles presentin said sample is greater than the purity of vesicles present in saidbiological sample. In certain embodiments, the agent is an antibody or alectin. Lectins useful for forming a vesicle-lectin complex aredescribed in U.S. Patent Application Publication No. 2012/0077263. Insome embodiments, the vesicle is an exosome, a microparticle, amicrovesicle, nanosomes, extracellular vesicles, or an ectosome. In someembodiments, the exosomes are neuron-derived exosomes, astrocyte-derivedexosomes, oliogodendrocyte-derived exosomes, or microglia-derivedexosomes. In some embodiments, multiple isolating or enriching steps areperformed. In certain aspects of the present embodiment, a firstisolating step is performed to isolate exosomes from a blood sample anda second isolating step is performed to isolate neural-derived exosomesfrom other exosomes. In other embodiments, the vesicle portion of thevesicle-agent complex is lysed using a lysis reagent and the proteinlevels of the lysed vesicle are assayed. In some embodiments, theantibody-vesicle complex is created on a solid phase. In yet otherembodiments, the methods further comprise releasing the vesicle from theantibody-vesicle complex. In certain embodiments, the solid phase isnon-magnetic beads, magnetic beads, agarose, or sepharose. In otherembodiments, the vesicle is released by exposing the antibody-vesiclecomplex to low pH between 3.5 and 1.5. In yet other embodiments, thereleased vesicle is neutralized by adding a high pH solution. In otherembodiments, the released vesicle is lysed by incubating the releasedvesicles with a lysis solution. In still other embodiments, the lysissolution contains inhibitors for proteases and phosphatases.

Neurodegenerative Disorders

The present invention provides methods for diagnosing or prognosing aneurodegenerative disorder in a subject, identifying a subject at riskof a neurodegenerative disorder, or prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder.

In some embodiments the neurodegenerative disorder is selected from thegroup consisting of: Alzheimer's disease (AD), vascular diseasedementia, frontotemporal dementia (FTD), corticobasal degeneration(CBD), progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease.

In some embodiments, the present invention enables a medicalpractitioner to diagnose or prognose one or more neurodegenerativedisorder in a subject. In other embodiments, the present inventionenables a medical practitioner to rule out or eliminate one or moreneurodegenerative diseases as a diagnostic possibility. In yet otherembodiments, the present invention enables a medical practitioner toidentify a subject at risk of developing a neurodegenerative disorder.In other embodiments, the present invention enables a medicalpractitioner to predict whether a subject will later develop aneurodegenerative disorder. In further embodiments the present inventionenables a medical practitioner to prescribe a therapeutic regimen orpredict benefit from therapy in a subject having a neurodegenerativedisorder.

Biomarkers

Biomarker levels are assayed in a biological sample obtained from asubject having or at-risk of having a neurodegenerative disorder (e.g.,Alzheimer's disease). In some embodiments, the biomarker isphosphorylated Tau, Aß1-42, TDP-43, α-synuclein, SOD-1, FUS, FKBP51,IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9,CD63, CD81, and CD171. In other embodiments, the phosphorylated tau isphosphorylated at one or more serine residues. In certain aspects of thepresent embodiment, the phosphorylated tau is phosphorylated on at leastone serine residue selected from the group consisting of Ser-199,Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, and Ser-422. Inother embodiments, the phosphorylated tau is phosphorylated at one ormore threonine residues. In certain aspects of the present embodiment,the phosphorylated tau is phosphorylated on at least one threonineresidue selected from the group consisting of Thr-153, Thr-181, Thr-205,and Thr-231. In yet other embodiments, the phosphorylated tau isphosphorylated at one or more tyrosine residues. In certain aspects ofthe present embodiment, the phosphorylated tau is phosphorylated on atleast one tyrosine residue selected from the group consisting of Tyr18,Tyr29, Tyr197, Tyr310, and Tyr 394. In other embodiments, phosphorylatedtau levels are determined or assayed using antibodies against one ormore phosphorylation sites. In certain aspects of the presentembodiment, the antibodies used in the present invention preferentiallybind to phosphorylated tau that is phosphorylated at one or more of thefollowing phosphorylation sites: Thr-153, Thr-181, Thr-205, Thr-231,Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396, Ser-422,Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. In other embodiments, thephosphorylated IRS-1 is P-S312-IRS-1 or P-panY-IRS-1.

In some embodiments, biomarker levels of the present invention aremeasured by determining the gene expression of the biomarker. In certainembodiments, gene expression changes are measured by determining theexpression level of one or more of the genes shown in Table 1. Incertain aspects, gene expression of the biomarker is determined usingPCR, microarray, or sequencing. In some embodiments, the expressionlevel of the biomarker is determined by measuring the mRNA or miRNAlevel of the biomarker.

One of ordinary skill in the art has several methods and devicesavailable for the detection and analysis of the markers of the instantinvention. With regard to polypeptides or proteins in patient testsamples, immunoassay devices and methods are often used. These devicesand methods can utilize labeled molecules in various sandwich,competitive, or non-competitive assay formats, to generate a signal thatis related to the presence or amount of an analyte of interest.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule.

Preferably the markers are analyzed using an immunoassay, although othermethods are well known to those skilled in the art (for example, themeasurement of marker RNA levels). The presence or amount of a marker isgenerally determined using antibodies specific for each marker anddetecting specific binding. Any suitable immunoassay may be utilized,for example, enzyme-linked immunoassays (ELISA), radioimmunoassay(RIAs), competitive binding assays, planar waveguide technology, and thelike. Specific immunological binding of the antibody to the marker canbe detected directly or indirectly. Direct labels include fluorescent orluminescent tags, metals, dyes, radionuclides, and the like, attached tothe antibody. Indirect labels include various enzymes well known in theart, such as alkaline phosphatase, horseradish peroxidase and the like.

The use of immobilized antibodies specific for the markers is alsocontemplated by the present invention. The antibodies could beimmobilized onto a variety of solid supports, such as magnetic orchromatographic matrix particles, the surface of an assay place (such asmicrotiter wells), pieces of a solid substrate material (such asplastic, nylon, paper), and the like. An assay strip could be preparedby coating the antibody or a plurality of antibodies in an array onsolid support. This strip could then be dipped into the test sample andthen processed quickly through washes and detection steps to generate ameasurable signal, such as a colored spot.

The analysis of a plurality of markers may be carried out separately orsimultaneously with one test sample. Several markers may be combinedinto one test for efficient processing of a multiple of samples. Inaddition, one skilled in the art would recognize the value of testingmultiple samples (for example, at successive time points) from the sameindividual. Such testing of serial samples will allow the identificationof changes in marker levels over time. Increases or decreases in markerlevels, as well as the absence of change in marker levels, would provideuseful information about the disease status that includes, but is notlimited to identifying the approximate time from onset of the event, thepresence and amount of salvageable tissue, the appropriateness of drugtherapies, the effectiveness of various therapies, identification of theseverity of the event, identification of the disease severity, andidentification of the patient's outcome, including risk of futureevents.

An assay consisting of a combination of the markers referenced in theinstant invention may be constructed to provide relevant informationrelated to differential diagnosis. Such a panel may be constructed using1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers.The analysis of a single marker or subsets of markers comprising alarger panel of markers could be carried out methods described withinthe instant invention to optimize clinical sensitivity or specificity invarious clinical settings. The ratio of P-S312-IRS-1 and P-panY-IRS-1 (Ror insulin resistance index) may be used to predict risk or diagnosis ofa neurodegenerative disorder.

The analysis of markers could be carried out in a variety of physicalformats as well. For example, the use of microtiter plates or automationcould be used to facilitate the processing of large numbers of testsamples. Alternatively, single sample formats could be developed tofacilitate immediate treatment and diagnosis in a timely fashion, forexample, in ambulatory transport or emergency room settings.Particularly useful physical formats comprise surfaces having aplurality of discrete, addressable locations for the detection of aplurality of different analytes. Such formats include proteinmicroarrays, or “protein chips” and capillary devices.

Biomarkers of the present invention serve an important role in the earlydetection and monitoring of neurodegenerative disorders (e.g.,Alzheimer's disease). Markers of such disorders are typically substancesfound in a bodily sample that can be measured. The measured amount cancorrelate to underlying disorder or disease pathophysiology, presence orabsence of a neurodegenerative disorder, probability of aneurodegenerative disorder in the future. In patients receivingtreatment for their condition the measured amount will also correlatewith responsiveness to therapy.

In some embodiments, the biomarker is measured by a method selected fromthe group consisting of immunohistochemistry, immunocytochemistry,immunofluorescence, immunoprecipitation, western blotting, and ELISA.

Clinical Assay Performance

The methods of the present invention may be used in clinical assays todiagnose or prognose a neurodegenerative disorder in a subject, identifya subject at risk of a neurodegenerative disorder, and/or forprescribing a therapeutic regimen or predicting benefit from therapy ina subject having a neurodegenerative disorder. Clinical assayperformance can be assessed by determining the assay's sensitivity,specificity, area under the ROC curve (AUC), accuracy, positivepredictive value (PPV), and negative predictive value (NPV). Disclosedherein are assays for diagnosing or prognosing a neurodegenerativedisorder in a subject, identifying a subject at risk of aneurodegenerative disorder, or for prescribing a therapeutic regimen orpredicting benefit from therapy in a subject having a neurodegenerativedisorder.

The clinical performance of the assay may be based on sensitivity. Thesensitivity of an assay of the present invention may be at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or100%. The clinical performance of the assay may be based on specificity.The specificity of an assay of the present invention may be at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%,or 100%. The clinical performance of the assay may be based on areaunder the ROC curve (AUC). The AUC of an assay of the present inventionmay be at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,or 0.95. The clinical performance of the assay may be based on accuracy.The accuracy of an assay of the present invention may be at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or100%.

Compositions

Compositions useful in the methods of the present invention includecompositions that specifically recognize a biomarker associated with aneurodegenerative disorder, wherein the biomarker is phosphorylated tau,Aß1-42, phosphorylated IRS, CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9,CD63, CD81, and CD171. In some embodiments, the composition enhances theactivity of at least one tau or IRS-1 phosphatase. In other embodiments,the composition decreases the activity of at least one tau or IRS-1kinase. In yet other embodiments, the composition is selected from thegroup consisting of a peptide, a nucleic acid, an antibody, and a smallmolecule.

In certain embodiments, the present invention relates to compositionsthat specifically detect a biomarker associated with a neurodegenerativedisorder. As detailed elsewhere herein, the present invention is basedupon the finding that phosphorylated tau, phosphorylated IRS-1, CTSD,LAMP1, UBP, HSP70, NSE, and NFL are specific biomarkers for AD and otherneurodegenerative disorders. In one embodiment, the compositions of theinvention specifically bind to and detect phosphorylated tau. In oneembodiment the compositions of the invention specifically bind to anddetect tau phosphorylated at one or more serine, threonine, or tyrosineresidues on tau. In some embodiments, the compositions of the inventionspecifically bind to tau phosphorylated at S396 (S396 tauphosphorylation). In some embodiments, the compositions of the inventionspecifically bind to tau phosphorylated at T181 (T181 tauphosphorylation). In still other embodiments, the compositions of theinvention specifically bind to tau phosphorylated at one or moreresidues selected from the group consisting of Thr-153, Thr-181,Thr-205, Thr-231, Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356,Ser-396, Ser-422, Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394. Thecomposition of the present invention can comprise an antibody, apeptide, a small molecule, a nucleic acid, and the like. In otherembodiments, the compositions of the present invention specifically bindto and detect phosphorylated IRS-1. In one embodiment the compositionsof the invention specifically bind to and detect P-S312-IRS-1 orP-panY-IRS-1. In other embodiments, the compositions of the presentinvention specifically bind to and detect CTSD, LAMP1, UBP, HSP70, NSE,NFL, CD9, CD63, CD81, and CD171.

In some embodiments, the composition comprises an antibody, where theantibody specifically binds to a biomarker or vesicles of the invention.The term “antibody” as used herein and further discussed below isintended to include fragments thereof which are also specificallyreactive with a biomarker or vesicle (e.g., exosome). Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described above for whole antibodies. Forexample, F(ab)2 fragments can be generated by treating antibody withpepsin. The resulting F(ab)2 fragment can be treated to reduce disulfidebridges to produce Fab fragments. Antigen-binding portions may also beproduced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies. Antigen-binding portions include, interalia, Fab, Fab′, F(ab′)2, Fv, dAb, and complementarity determiningregion (CDR) fragments, single-chain antibodies (scFv), single domainantibodies, bispecific antibodies, chimeric antibodies, humanizedantibodies, diabodies and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. In certain embodiments, the antibody furthercomprises a label attached thereto and able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain embodiments, an antibody of the invention is a monoclonalantibody, and in certain embodiments, the invention makes availablemethods for generating novel antibodies that specifically bind thebiomarker or the exosome of the invention. For example, a method forgenerating a monoclonal antibody that specifically binds a biomarker orexosome, may comprise administering to a mouse an amount of animmunogenic composition comprising the biomarker or exosome, or fragmentthereof, effective to stimulate a detectable immune response, obtainingantibody-producing cells (e.g., cells from the spleen) from the mouseand fusing the antibody-producing cells with myeloma cells to obtainantibody-producing hybridomas, and testing the antibody-producinghybridomas to identify a hybridoma that produces a monocolonal antibodythat binds specifically to the biomarker or exosome. Once obtained, ahybridoma can be propagated in a cell culture, optionally in cultureconditions where the hybridoma-derived cells produce the monoclonalantibody that binds specifically to the biomarker or exosome. Themonoclonal antibody may be purified from the cell culture.

The term “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., a biomarker or exosome) and other antigens that are notof interest. In certain methods employing the antibody, such astherapeutic applications, a higher degree of specificity in binding maybe desirable. Monoclonal antibodies generally have a greater tendency(as compared to polyclonal antibodies) to discriminate effectivelybetween the desired antigens and cross-reacting polypeptides. Onecharacteristic that influences the specificity of an antibody:antigeninteraction is the affinity of the antibody for the antigen. Althoughthe desired specificity may be reached with a range of differentaffinities, generally preferred antibodies will have an affinity (adissociation constant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

Antibodies can be generated to bind specifically to an epitope of anexosome or a biomarker of the present invention, including, for example,neuron-derived exosome, phosphorylated Tau, Aß1-42, phosphorylatedIRS-1, CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9, CD63, CD81, and CD171.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. A variety of different techniques are available fortesting interaction between antibodies and antigens to identifyparticularly desirable antibodies. Such techniques include ELISAs,surface plasmon resonance binding assays (e.g., the Biacore bindingassay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., theparamagnetic bead system of IGEN International, Inc., Gaithersburg,Md.), western blots, immunoprecipitation assays, immunocytochemistry,and immunohistochemistry.

In some embodiments, the present invention relates to compositions usedfor treating or preventing a neurodegenerative disorder. As detailedelsewhere herein, the present invention is based upon the findings thattau phosphorylation is implicated in the pathology of a variety ofneurodegenerative disorders, such as, for example, Alzheimer's disease.Therefore, in one embodiment, the present invention providescompositions that prevent tau phosphorylation. In one embodiment, thecompositions prevent tau phosphorylation at one or more serine residueson tau. In other embodiments, the compositions prevent tauphosphorylation at one or more threonine residues on tau. In otherembodiments, the compositions prevent tau phosphorylation at one or moretyrosine residues on tau. In another embodiment, the present inventionprovides compositions that reduce tau phosphorylation. In oneembodiment, the compositions reduce tau phosphorylation at one or moreserine, threonine, and/or tyrosine residues on tau. In yet otherembodiments, the compositions reduce tau phosphorylation at one or moreresidues selected from the group consisting of Thr-153, Thr-181,Thr-205, Thr-231, Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356,Ser-396, Ser-422, Tyr18, Tyr29, Tyr197, Tyr310, and Tyr 394.

In some embodiments, the present invention relates to compositions usedfor treating or preventing a neurodegenerative disorder. As detailedelsewhere herein, the present invention is based upon the findings thatIRS-1 phosphorylation is implicated in the pathology of a variety ofneurodegenerative disorders, such as, for example, Alzheimer's disease.Therefore, in one embodiment, the present invention providescompositions that prevent IRS-1 phosphorylation. In another embodiment,the present invention provides compositions that reduce IRS-1phosphorylation.

Compositions useful for preventing and/or reducing tau or IRS-1phosphorylation include proteins, peptides, nucleic acids, smallmolecules, and the like.

Methods of Treatment

The present invention provides methods of treating a neurodegenerativedisorder in a subject, comprising administering to the subject aneffective amount of a composition, wherein the composition reduces thelevel of tau and/or IRS-1 phosphorylation. In other embodiments, thecomposition enhances the activity of at least one tau or IRS-1phosphatase. In yet other embodiments, the composition reduces theactivity of at least one tau or IRS-1 kinase. In other embodiments, thecomposition is selected from the group consisting of a peptide, anucleic acid, an antibody, and a small molecule. In other embodiments,the present invention provides methods of treating a neurodegenerativedisorder in a subject, comprising administering to the subject aneffective amount of a composition, wherein the composition reduces thelevel of CTSD, LAMP1, or UBP. In yet other embodiments, the presentinvention provides methods of treating a neurodegenerative disorder in asubject, comprising administering to the subject an effective amount ofa composition, wherein the composition increases the level of HSP70. Inother embodiments, the present invention provides methods of treating aneurodegenerative disorder in a subject, comprising administering to thesubject an effective amount of a composition, wherein the compositionnormalizes the level of phosphorylated tau, Aß1-42, phosphorylatedIRS-1, CTSD, LAMP1, UBP, HSP70, NSE NFL, CD9, CD63, CD81, and CD171 to areference level.

Kits

Another aspect of the invention encompasses kits for detecting ormonitoring a neurodegenerative disorder in a subject. A variety of kitshaving different components are contemplated by the current invention.Generally speaking, the kit will include the means for quantifying oneor more biomarkers in a subject. In another embodiment, the kit willinclude means for collecting a biological sample, means for quantifyingone or more biomarkers in the biological sample, and instructions foruse of the kit contents. In certain embodiments, the kit comprises ameans for enriching or isolating exosomes in a biological sample. Infurther aspects, the means for enriching or isolating exosomes comprisesreagents necessary to enrich or isolate exosomes from a biologicalsample. In certain aspects, the kit comprises a means for quantifyingthe amount of a biomarker. In further aspects, the means for quantifyingthe amount of a biomarker comprises reagents necessary to detect theamount of a biomarker.

TABLE 1 Entrez Gene Gene Name Location microtubule-associated MAPTChromosome 17, NC_000017.11 protein tau (45894336..46028334) amyloidbeta (A4) APP Chromosome 21, NC_000021.9 precursor protein(25880550..26171128) TAR DNA-binding TARDBP Chromosome 1, NC_000001.11protein 43 (11012622..11025492) Alpha-synuclein SNCA Chromosome 4,NC_000004.12 (89724099..89838296) superoxide SOD1 Chromosome 21,NC_000021.9 dismutase 1 (31659622..31668931) FUS RNA FUS Chromosome 16,NC_000016.10 binding protein (31180110..31194871) FK506 binding FKBP51Chromosome 6, NC_000006.12 protein 51 (35573585..35728583) insulinreceptor IRS1 Chromosome 2, NC_000002.12 substrate 1(226731317..226799829) cathepsin D CTSD Chromosome 11, NC_000011.10(1752752..1763992) lysosomal-associated LAMP1 Chromosome 13,NC_000013.11 membrane protein 1 (113297154..113323426) ubiquitin B UBBChromosome 17, NC_000017.11 (16380793..16382745) Ubiquitin C UBCChromosome 12, NC_000012.12 (124911646..124915041) ubiquitin A-52residue UBA52 Chromosome 19, NC_000019.10 ribosomal protein(18563766..18577460) fusion product 1 ribosomal protein RPS27AChromosome 2, NC_000002.12 S27a (55231903..55235853) heat shock 70kDaHSPA1A Chromosome 6, NC_000006.12 protein 1A (31815514..31817942) heatshock 70kDa HSPA4 Chromosome 5, NC_000005.10 protein 4(133051970..133105017) enolase 2 (gamma, ENO2 Chromosome 12,NC_000012.12 neuronal) (6914450..6923696) neurofilament, light NEFLChromosome 8, NC_000008.11 polypeptide (24950955..24956869, complement)

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

EXAMPLES

The invention will be further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.These examples are provided solely to illustrate the claimed invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Example 1 Neuron-Derived Serum Exosomal Phosphorylated Tau and Aß1-42Levels in Human Subjects with Alzheimer's Disease

Levels of Phosphorylated Tau and Aß1-42 protein were assayed in humansubjects with Alzheimer's disease (AD) as follows. Ten milliliters ofvenous blood were collected from control subjects (n=20) and subjectswith AD (n=20). A first blood sample was collected at a time when ADsubjects were cognitively intact. A second blood sample was collectedafter AD subjects had developed probable AD. The diagnosis of AD wasestablished by standard clinical and laboratory criteria.

For blood collection, 10 ml of venous blood were taken from each subjectand held at 37° C. for 45 min and centrifuged at 1,000×g for 20 min at4° C. to obtain serum for storage in 0.5 ml aliquots at −80° C. Next,0.5 ml of each serum sample was mixed with 0.5 ml of calcium- andmagnesium-free Dulbecco's balanced salt solution (DBS) containing twicethe recommended concentrations of protease inhibitor cocktail (RocheApplied Sciences, Inc., Indianapolis, Ind.) phosphatase inhibitorcocktail (Pierce Halt, Thermo Scientific, Inc., Rockford, Ill.), andincubated for 15 minutes at room temperature.

For plasma, 0.5 ml received 0.1 ml of thromboplastin-D (FisherScientific, Inc., Hanover Park, Ill.) followed by incubation at roomtemperature for 30 min and centrifugation at 1,500×g for 5 min. Then 0.4ml of DBS containing 2.5-times the recommended concentrations ofprotease and phosphatase inhibitor cocktails were added to eachsupernatant.

These serum and plasma supernatants were mixed thoroughly with 252 μl ofExoQuick exosome precipitation solution (EXOQ; System Biosciences, Inc.,Mountainview, Calif.), and the mixture incubated for 1 hour at 4° C. Theresulting exosome suspensions were centrifuged at 1,500×g for 30 min at4° C., the supernatants removed and each pellet of exosomes resuspendedin 250 μl of DBS with the cocktails of protease and phosphataseinhibitors for immunochemical enrichment of exosomes from a neuralsource. Next, each sample received 2 μg of mouse anti-human NCAMantibody (ERIC 1, sc-106, Santa Cruz Biotechnology, Santa Cruz, Calif.),that had been previously biotinylated with the EZ-Link sulfo-NHS-biotinsystem (Thermo Scientific, Inc.). After 2 hr at 4° C., 20 μl ofstreptavidin-agarose resin (Thermo Scientific, Inc.) was added to eachsample followed by incubation for 1 hr at 4° C. with rocking. Thesamples then were centrifuged at 200 g for 10 min at 4° C., thesupernates removed and each pellet resuspended in 250 μl of ELISAbinding buffer (System Biosciences, Inc.) with the cocktails of proteaseand phosphatase inhibitors.

Neuron-derived exosomal proteins were quantified by ELISA kits for humanCD81 (System Biosciences, Inc. and Hölzel Diagnostika, Cologne, Germany)with verification of antigen with human purified recombinant CD81antigen (Origene Technologies, Inc., Rockville, Md.), human Aß1-42,total human tau and human phosphorylated tau [pS396] (LifeTechnologies/Invitrogen, Camarillo, Calif.), and human phosphorylatedtau [pT181] (Innogenetics, Inc., Alpharetta, Ga.) according to thesuppliers' directions.

As shown in Table 2 below, neuron-derived serum exosomal phosphorylatedTau (pS396) levels were significantly increased in subjects with ADcompared to control subject serum exosomal levels. Similarly, serumexosomal Aß1-42 levels were significantly increased in subjects with ADcompared to control levels. Serum exosomal total Tau levels were notsignificantly different between AD and control subjects.

TABLE 2 Group Total Tau (pg/ml) Aβ1-42 (pg/ml) pS396 Tau (pg/ml) AD (n =20) 290 ± 160 14.28 ± 4.61⁺ 13.46 ± 10.38* Control (n = 20) 298 ± 125 9.61 ± 2.92 1.46 ± 0.35  Mean ± SD; *p < 0.0001 compared to Control, ⁺p= 0.0005 by a two-tailed unpaired t test.

These results showed that neuron-derived serum exosomal levels of Aß1-42and phosphorylated Tau are useful for identifying subjects with AD.These results further indicated that methods and compositions of thepresent invention are useful for diagnosing Alzheimer's disease andother neurodegenerative disorders.

A second series of experiments were carried out to determine the levelsof phosphorylated Tau and Aß1-42 protein in human subjects with earlyAlzheimer's disease (i.e., minimal cognitive impairment, MCI) and lateAlzheimer's disease. Ten milliliters of venous blood were collected fromsubjects with early AD (n=10) and subjects with late AD (n=8). Bloodsamples were processed as described above and neuron-derived exosomalprotein levels for Aß1-42, Tau, and phosphorylated Tau were quantifiedusing the ELISA kits described above.

As shown in Table 3 below, neuron-derived serum exosomal phosphorylatedTau (pS396) levels were similar in early AD and late AD. Serum exosomalAß1-42 levels were also similar in early AD and late AD.

TABLE 3 Group Total Tau (pg/ml) Aβ1-42 (pg/ml) pS396 Tau (pg/ml) EarlyAD (n = 10) 315 ± 152 13.76 ± 4.27 13.81 ± 9.30 Late AD (n = 8) 305 ±96  13.44 ± 3.11  13.83 ± 13.30 Values are mean ± SD

These results showed that neuron-derived serum exosomal levels of Aß1-42and phosphorylated Tau are useful for identifying subjects with early ADor late AD. These results further indicated that methods andcompositions of the present invention are useful for diagnosingAlzheimer's disease and other neurodegenerative disorders.

Example 2 Neuron-Derived Serum Exosomal Phosphorylated Tau Levels inHuman Subjects with Alzheimer's Disease

Neuron-derived exosomal levels of phosphorylated Tau protein are assayedin human subjects with Alzheimer's disease (AD) as follows. Bloodsamples are collected from human subjects with AD and processed asdescribed in Example 1 above except that, following neuron-derivedexosome enrichment, the samples are assayed for the followingphosphorylated Tau proteins: pSer-199 Tau, pSer-202 Tau, pSer-214 Tau,pSer-235 Tau, pSer-262 Tau, pSer-356 Tau, pSer-422 Tau, pThr-153 Tau,pThr-181 Tau, pThr-205 Tau, or pThr-231 Tau.

Neuron-derived exosomal phosphorylated Tau levels are significantlyincreased compared to control levels. These results show thatneuron-derived serum exosomal levels of phosphorylated Tau are usefulfor identifying subjects with AD. These results further indicate thatmethods and compositions of the present invention are useful fordiagnosing Alzheimer's disease and other neurodegenerative disorders.

Example 3 Neuron-Derived Serum Exosomal Protein Levels in Human Subjectswith Multiple Sclerosis

Levels of Phosphorylated Tau protein are assayed in human subjects withMultiple Sclerosis (MS) as follows. Blood samples are collected fromhuman subjects with MS and processed as described in Example 1 aboveexcept that, following neuron-derived exosome enrichment, the samplesare assayed for the following exosomal proteins: pSer-199 Tau, pSer-202Tau, pSer-214 Tau, pSer-235 Tau, pSer-262 Tau, pSer-356 Tau, pSer-396Tau, pSer-422 Tau, pThr-153 Tau, pThr-181 Tau, pThr-205 Tau, or pThr-231Tau.

Neuron-derived exosomal phosphorylated Tau levels are significantlyincreased compared to control levels. These results show thatneuron-derived serum exosomal levels of phosphorylated Tau are usefulfor identifying subjects with MS. These results further indicate thatmethods and compositions of the present invention are useful fordiagnosing multiple sclerosis and other neurodegenerative disorders.

Example 4 Neuron-Derived Serum Exosomal Protein Levels in Human Subjectswith Frontotemporal Dementia

Levels of Phosphorylated Tau protein are assayed in human subjects withfrontotemporal dementia (FTD) as follows. Blood samples are collectedfrom human subjects with FTD and processed as described in Example 1above except that, following neuron-derived exosome enrichment, thesamples are assayed for the following exosomal proteins: pSer-199 Tau,pSer-202 Tau, pSer-214 Tau, pSer-235 Tau, pSer-262 Tau, pSer-356 Tau,pSer-396 Tau, pSer-422 Tau, pThr-153 Tau, pThr-181 Tau, pThr-205 Tau, orpThr-231 Tau.

Neuron-derived exosomal phosphorylated Tau levels are significantlyincreased compared to control levels. These results show thatneuron-derived serum exosomal levels of phosphorylated Tau are usefulfor identifying subjects with FTD. These results further indicate thatmethods and compositions of the present invention are useful fordiagnosing frontotemporal dementia and other neurodegenerativedisorders.

Example 5 Neuron-Derived Serum Exosomal Protein Levels PredictAlzheimer's Disease in Human Subjects

Neuron-derived exosomal levels of phosphorylated Tau protein are assayedin human subjects to predict Alzheimer's disease (AD) as follows (e.g.,identify subjects at-risk of developing Alzheimer's disease). Bloodsamples are collected from human subjects prior to diagnosis of AD(e.g., prior to AD onset) and again after a diagnosis of Alzheimer'sdisease (e.g., after developing overt dementia). Blood samples areprocessed as described in Example 1 above except that, followingneuron-derived exosome enrichment, the samples are assayed for thefollowing exosomal proteins: pSer-199 Tau, pSer-202 Tau, pSer-214 Tau,pSer-235 Tau, pSer-262 Tau, pSer-356 Tau, pSer-396 Tau, pSer-422 Tau,pThr-153 Tau, pThr-181 Tau, pThr-205 Tau, or pThr-231 Tau.

Neuron-derived exosomal phosphorylated Tau levels are significantlyincreased compared to control levels in blood samples taken before andafter AD diagnosis (i.e., increased neuron-derived exosomalphosphorylated Tau levels before AD onset and after developing overtdementia). These results show that neuron-derived serum exosomal levelsof phosphorylated Tau are useful for predicting whether a subject willdevelop AD. These results further indicate that methods and compositionsof the present invention are useful for prognosing and diagnosingAlzheimer's disease and other neurodegenerative disorders.

Example 6 Neuron-Derived Serum Exosomal Phosphorylated Tau, Total Tau,and Aß1-42 Levels are Increased in Human Subjects with Alzheimer'sDisease

Exosomal levels of total Tau, phosphorylated Tau (P-T181 and P-S396),and Aß1-42 protein levels were assayed in human subjects withAlzheimer's disease (AD) as follows. Ten milliliters of venous bloodwere collected from subjects with AD (n=57) and control subjects (n=57).Blood samples were processed and exosomes were isolated as described inExample 1 above.

Exosome proteins were quantified by ELISA kits for human Aß1-42, humantotal tau and human P-S396-tau (Life Technologies/Invitrogen, Camarillo,Calif.), human P-T181-tau (Innogenetics Division of Fujirebio US, Inc.,Alpharetta, Ga.) and human CD81 (Hölzel Diagnostika-Cusabio, Cologne,Germany) with verification of the CD81 antigen standard curve usinghuman purified recombinant CD81 antigen (Origene Technologies, Inc.,Rockville, MD), according to suppliers' directions.

Separate discriminant classifier analyses were conducted to define thebest simple linear models for comparing AD subjects with controlsubjects. Two discriminant analyses considered all variables and wereperformed step-wise. Final models retained only variables with a minimumpartial F of 3.84 to enter and 2.71 to remove. Prior probabilities wereconsidered equal for all groups. Fisher Function Coefficients and withingroup covariances were computed. Receiver operating characteristics(ROC) analyses were conducted under the non-parametric distributionassumption for Standard Error of Area to determine the performance ofthe models for discriminating AD subjects from control subjects.Discriminant and ROC analyses were conducted with SPSS v21.0 (IBM).

As shown in Table 4 below, neuron-derived serum exosomal total tau,P-T181-tau, P-S396-tau, and Aß31-42 levels were significantly increasedin subjects with AD compared to control subject serum exosomal levels.

TABLE 4 Group Total Tau (pg/ml) pT181 Tau (pg/ml) pS396 Tau (pg/ml)Aβ1-42 (pg/ml) AD (n = 57) 191 ± 12.3⁺ 106 ± 6.10* 25.4 ± 2.25*  18.5 ±2.97* Control (n = 57) 130 ± 11.9  16.9 ± 1.89   3.88 ± 0.26  0.83 ±0.13 Mean ± SD; *p < 0.0001 compared to Control, ⁺p = 0.0005 by anunpaired t test.

Step-wise discriminant analyses resulted in a model progressivelyincorporating P-T181-tau, P-S396-tau and Aß1-42, but not total tau,which produced a Wilk's Lambda of 0.229 and an exact F of 119 (p<0.001).The final model correctly classified 96.4% of AD patients. The AreaUnder the Curve (AUC) for the final model from the ROC analysis was0.999 and individual AUC values for the individual proteins were 0.991,0.988, 0.987 and 0.731, respectively, for P-T181-tau, P-S396-tau, Aß1-42and total tau.

These results showed that neuron-derived serum exosomal levels of totalTau, P-T181-tau, P-S396-tau, and Aß1-42 are increased in subjects withAlzheimer's disease and are useful for identifying subjects withAlzheimer's disease. These results further showed that assays andmethods of the present invention correctly classified subjects withAlzheimer's disease. These results further indicated that methods andcompositions of the present invention are useful for diagnosingAlzheimer's disease and other neurodegenerative disorders.

Example 7 Neuron-Derived Serum Exosomal Phosphorylated Tau, Total Tau,and Aß1-42 Levels are Increased in Human Subjects with FrontotemporalDisease

Exosomal levels of total Tau, phosphorylated Tau (P-T181 and P-S396),and Aß1-42 protein levels were assayed in human subjects withfrontotemporal disease (FTD) as follows. Ten milliliters of venous bloodwere collected from subjects with FTD (n=16) and control subjects(n=16). Blood samples were processed and exosomes were isolated asdescribed in Example 1 above.

Exosome proteins were quantified by ELISA kits for human Aß1-42, humantotal tau and human P-5396-tau (Life Technologies/Invitrogen, Camarillo,Calif.), human P-T181-tau (Innogenetics Division of Fujirebio US, Inc.,Alpharetta, Ga.) and human CD81 (Hölzel Diagnostika-Cusabio, Cologne,Germany) with verification of the CD81 antigen standard curve usinghuman purified recombinant CD81 antigen (Origene Technologies, Inc.,Rockville, Md.), according to suppliers' directions.

Separate discriminant classifier analyses were conducted to define thebest simple linear models for comparing FTD subjects with controlsubjects. Two discriminant analyses considered all variables and wereperformed step-wise. Final models retained only variables with a minimumpartial F of 3.84 to enter and 2.71 to remove. Prior probabilities wereconsidered equal for all groups. Fisher Function Coefficients and withingroup covariances were computed. Receiver operating characteristics(ROC) analyses were conducted under the non-parametric distributionassumption for Standard Error of Area to determine the performance ofthe models for discriminating FTD subjects from control subjects.Discriminant and ROC analyses were conducted with SPSS v21.0 (IBM).

As shown in Table 5 below, neuron-derived serum exosomal P-T181-tau andAß1-42 levels were significantly increased in subjects with FTD comparedto control subject serum exosomal levels.

TABLE 5 Group Total Tau (pg/ml) pT181 Tau (pg/ml) pS396 Tau (pg/ml)Aβ1-42 (pg/ml) FTD (n = 16) 135 ± 15.8 82.6 ± 9.20* 2.13 ± 0.33 7.54 ±1.01* Control (n = 16) 148 ± 30.1 9.32 ± 2.86  3.13 ± 0.46 0.76 ± 0.35 Mean ± SD; *p < 0.0001 compared to Control by an unpaired t test.

In a step-wise discriminant analysis, P-T181-tau attained a Wilk'sLambda value of 0.324 and an exact F of 62.5 (p<0.001). In a finalmodel, exosomal P-T181-tau correctly classified 87.5% of FTD patientscontrasted with control subjects (75% of FTD and 100% of control). Forthe final model from the ROC analysis, AUC for P-T181-tau was 0.992 andfor Aß1-42 was 0.969.

These results showed that neuron-derived serum exosomal levels ofP-T181-tau and Aß1-42 are increased in subjects with frontotemporaldisease and are useful for identifying subjects with frontotemporaldisease. These results further showed that assays and methods of thepresent invention correctly classified subjects with frontotemporaldisease. These results further indicated that methods and compositionsof the present invention are useful for diagnosing frontotemporaldisease and other neurodegenerative disorders.

Example 8 Neuron-Derived Serum Exosomal Phosphorylated Tau and Aß1-42Levels Predict Development of Alzheimer's Disease in Human Subjects

Exosomal levels of total Tau, phosphorylated Tau (P-T181 and P-S396),and A131-42 protein levels were assayed in human subjects as follows.Ten milliliters of venous blood were collected from subjects (n=24) attwo time-points: the first at one to ten years before the subjects'diagnosis of Alzheimer's disease (Alzheimer's preclinical, AP) and thesecond at the time of initial diagnosis of Alzheimer's disease (AD).Venous blood samples were also collected from control subjects (n=24).Blood samples were processed and exosomes were isolated as described inExample 1 above.

Exosome proteins were quantified by ELISA kits for human Aß1-42, humantotal tau and human P-S396-tau (Life Technologies/Invitrogen, Camarillo,Calif.), human P-T181-tau (Innogenetics Division of Fujirebio US, Inc.,Alpharetta, Ga.) and human CD81 (Hölzel Diagnostika-Cusabio, Cologne,Germany) with verification of the CD81 antigen standard curve usinghuman purified recombinant CD81 antigen (Origene Technologies, Inc.,Rockville, Md.), according to suppliers' directions.

As shown in Table 6 below, neuron-derived serum exosomal P-T181-tau,P-S396-tau, and Aß1-42 levels were significantly increased in subjectswith AD compared to control subject serum exosomal levels. Further,neuron-derived serum exosomal P-T181-tau and P-S396-tau levels weresignificantly increased in subjects as early as 10 years before clinicaldiagnosis of AD (see Table 6). For Aß1-42, mean levels for the AD and APgroups both were significantly higher than control subject levels, andthe mean AD level also was significantly higher than that of the APgroup (see Table 6).

TABLE 6 Group Total Tau (pg/ml) pT181 Tau (pg/ml) pS396 Tau (pg/ml)Aβ1-42 (pg/ml) AD (n = 24) 165 ± 15.8 91.1 ± 4.42* 25.2 ± 1.85* 14.5 ±1.41* AP (n = 24) 154 ± 13.6 85.7 ± 3.75* 19.2 ± 2.00*  6.64 ± 0.58*^(#)Control (n = 16)  148 ± 116.5 35.6 ± 3.49  4.72 ± 0.64  1.51 ± 0.52 Mean ± SEM; *p < 0.0001 compared to Control by paired t test; ^(#)p <0.001 compared to AD by paired t test.

These results showed that neuron-derived serum exosomal levels ofP-T181-tau, P-S396-tau, and Aß1-42 are increased in subjects withAlzheimer's disease and are useful for identifying subjects withAlzheimer's disease. These results also showed that neuron-derived serumexosomal levels of P-T181-tau, P-S396-tau, and Aß1-42 are increased insubjects as early as 10 years before clinical diagnosis of Alzheimer'sdisease. These results further showed that assays and methods of thepresent invention are useful for identifying a subject at risk of aneurodegenerative disorder (e.g., Alzheimer's disease). Additionally,these results showed that the assays and methods of the presentinvention may be useful for early detection and determining theprogression of Alzheimer's disease. These results further indicated thatmethods and compositions of the present invention are useful fordiagnosing Alzheimer's disease and other neurodegenerative disorders.

Example 9 Neuron-Derived Plasma Exosomal Total IRS-1 and PhosphorylatedIRS-1 Levels in Human Subjects with a Neurodegenerative Disorder orDiabetes

Exosomal levels of total IRS-1 and phosphorylated IRS-1 (P-S312-IRS-1and P-panY-IRS-1) protein levels were assayed in human subjects asfollows. Thirty milliliters of venous blood were collected from 26subjects with AD, 20 subjects with type 2 diabetes mellitus (DM2), 16subjects with FTD, and matched control subjects. Blood samples wereincubated for 10 min at room temperature and centrifuged for 15 min at1500g. Plasma was aspirated and stored in aliquots at −80° C.

One-half ml of plasma was incubated with 0.15 ml of thromboplastin-D(Fisher Scientific, Inc., Hanover Park, Ill.) at room temperature for 1hour, followed by addition of 0.35 ml of calcium- and magnesium-freeDulbecco's balanced salt solution (DBS⁻²) with three-times the suggestedfinal concentrations of protease inhibitor cocktail (Roche AppliedSciences, Inc., Indianapolis, Ind.) and phosphatase inhibitor cocktail(Pierce Halt, Thermo Scientific, Inc., Rockford, Ill.). Aftercentrifugation at 1,500 g for 20 min, supernates were mixed with 252 μlof ExoQuick exosome precipitation solution (EXOQ; System Biosciences,Inc., Mountainview, Calif.), and incubated for 1 hour at 4° C. Resultantexosome suspensions were centrifuged at 1,500 g for 30 min at 4° C. andeach pellet was re-suspended in 250 μl of distilled water with inhibitorcocktails by vortex-mixing after a −80° C. freeze-thaw cycle beforeimmunochemical enrichment of exosomes from neural sources.

Each sample was incubated for 1 hour at 4° C. with 1 μg of mouseanti-human CD171 (L1CAM neural adhesion protein) biotinylated antibody(clone 5G3, eBioscience, San Diego, Calif.) in 50 μL of 3% BSA (1:3.33dilution of Blocker BSA 10% solution in PBS, Thermo Scientific, Inc.)with 50 μL of 3% BSA and incubation for 30 min at 4° C. Aftercentrifugation at 200 g for 10 min at 4° C. and removal of thesupernate, each pellet was resuspended in 0.5 ml of M-PER mammalianprotein extraction reagent (Thermo Scientific, Inc.), that had beenadjusted to pH 8.0 with 1 M Tris-HCl (pH 8.6) and contained thecocktails of protease and phosphatase inhibitors. These suspensions wereincubated at 37° C. for 20 min and vortex-mixed for 15 sec beforestorage at −80° C. until use in ELISAs.

Exosome proteins were quantified by ELISA kits for human P-S312-IRS-1(Life Technologies Corp., Carlsbad, Calif.), human P-panY-IRS-1 (CellSignaling Technology, Danvers, Mass.), human total IRS-1 (AMSBIO, LLC,Cambridge, Mass.), and tetraspanning exosome marker human CD81 (HölzelDiagnostika-Cusabio, Cologne, Germany) with verification of the CD81antigen standard curve using human purified recombinant CD81 antigen(Origene Technologies, Inc., Rockville, Md.), according to suppliers'directions. The mean value for all determinations of CD81 in each assaygroup was set at 1.00 and the relative values for each sample used tonormalize their recovery. R, the insulin resistance index, was definedas the ratio of P-S312-IRS-1 and P-panY-IRS-1.

The statistical significance of differences between group means forpatients with AD or FTD or DM2, and between each patient group and theirrespective matched control groups was determined with an unpaired t testincluding a Bonferroni correction in the interpretation (GraphPad Prism6, La Jolla, Calif.). Separate discriminant classifier analyses wereconducted to define the best simple linear models for comparing group ADwith its control group (AC), FTD with its control group (FTC) and DM2with its control group (DC). The discriminant analyses were performedstep-wise with the Wilks' Lambda method. In each step, only variableswith a minimum partial F of 3.84 to enter and 2.71 to remove wereretained. Prior probabilities were considered equal for all groups.Fisher Function Coefficients and within group covariances also werecomputed. Receiver operating characteristics (ROC) analyses wereconducted under the non-parametric distribution assumption for StandardError of Area to determine the performance of the models fordiscriminating AD from AC, FTD from FTC, DM2 from DC, and AD from DM2and FTD. Discriminant and ROC analyses were conducted with SPSS v21.0(IBM). To assess significance of associations between CSF biomarkers inAD patients and IRS-1 proteins in neurally-enriched exosomes, wecomputed zero-order Pearson's correlations and partial correlations(controlling for age and sex). For longitudinal analyses, thesignificance of differences between serial values for AD patients takenbefore and after onset of aMCI or dementia was calculated with a pairedt test (GraphPad).

As shown in Table 7 below, neuron-derived serum exosomal P-S312-IRS-1levels were significantly increased in subjects with AD, DM2, and FTDcompared to their respective control subject serum exosomal levels. Theratio of insulin resistance index (R) was significantly increased insubjects with AD, DM2, and FTD compared to their respective controls(see Table 7). For P-panY-IRS-1, mean levels for the AD and DM2 groupsboth were significantly higher than control subject levels (see Table7).

TABLE 7 Insulin IRS-1 P-S312-IRS-1 P-panY-IRS-1 Resistance Group (pg/ml)(U/ml) (AU) Index (R) AD (n = 26) 5.04 ± 0.10 7.72 ± 0.45* 0.085 ±0.003* 92.2 ± 5.34* AC (n = 26) 5.45 ± 0.11 3.94 ± 0.30  0.204 ± 0.005 19.4 ± 1.44  DM2 (n = 20) 5.59 ± 0.12 5.48 ± 0.11* 0.143 ± 0.015* 50.4 ±6.93* DC (n = 20) 5.34 ± 0.12 3.24 ± 0.14  0.199 ± 0.005  16.4 ± 0.67 FTD (n = 16) 5.54 ± 0.15 6.12 ± 0.37* 0.226 ± 0.087  29.5 ± 2.08* FTC (n= 16) 5.30 ± 0.14 4.19 ± 0.18  0.215 ± 0.010  19.9 ± 1.30  Mean ± SEM;*p < 0.0001 compared to Control by paired t test.

Step-wise discriminant analysis of AD and AC data resulted in a modelthat incorporated first P-panY-IRS-1 and second P-S312-IRS-1. The finalmodel achieved a Wilks' Lambda of 0.105 and an exact F of 207.9(P<0.001), and correctly classified 100% of AD patients and AC subjects.In the ROC analysis of classification of the AD and AC groups,individual subject scores from the final model achieved an area underthe curve (AUC) of 1 (asymptotic significance<0.001). The AUC valueswere 0.722, 0.862 and 0.999, respectively, for total IRS-1,P-S312-IRS-1, and P-panY-IRS-1. Step-wise discriminant analysis of datathat distinguished DM2 from DC resulted in a model that incorporatedfirst P-panY-IRS-1 and second P-S312-IRS-1. The final model achieved aWilks' Lambda of 0.168 and an exact F of 91.8 (P<0.001) and correctlyclassified 97.5% of DM2 patients and DC subjects. In ROC analysis ofclassification of the DM2 and DC groups, individual subject scores fromthe final model achieved an AUC of 1. The AUC values were 0.741(asymptotic significance=0.009) and 0.999 (asymptoticsignificance<0.001), respectively, for P-panY-IRS-1 and P-S312-IRS-1.Step-wise discriminant analysis of data that distinguished FTD from FTCresulted in a model that incorporated P-5312-IRS-1. The final modelachieved a Wilks' Lambda of 0.576 and an exact F of 22.1 (P<0.001) andcorrectly classified 84% of FTD patients and FTC subjects. In ROCanalysis of classification of the FTD and FTC groups, P-S312-IRS-1achieved an AUC of 0.928 (asymptotic significance<0.001).

These results showed that neuron-derived serum exosomal levels ofP-S312-IRS-1, P-panY-IRS-1, and the ratio of insulin resistance index(R) are increased in subjects with AD, DM2, and FTD and are useful foridentifying subjects with AD, DM2, and FTD. These results furtherindicated that methods and compositions of the present invention areuseful for diagnosing Alzheimer's disease, diabetes, frontotemporaldementia and other neurodegenerative disorders.

Example 10 Neuron-Derived Serum Exosomal Phosphorylated IRS-1 Levels andan Insulin Resistance Index Predict Development of Alzheimer's Diseasein Human Subjects

Exosomal levels of total IRS-1 and phosphorylated IRS-1 (P-S312-IRS-1and P-panY-IRS-1) protein levels were assayed in human subjects asfollows. Ten milliliters of venous blood were collected from subjects(n=22) at two time-points: the first at one to ten years before thesubjects' diagnosis of Alzheimer's disease (Alzheimer's preclinical, AP)and the second at the time of initial diagnosis of Alzheimer's disease(AD). Venous blood samples were also collected from control subjects(n=22). Blood samples were processed and exosomes were isolated asdescribed in Example 9 above.

As shown in Table 8 below, neuron-derived serum exosomal serum exosomalP-S312-IRS-1, P-panY-IRS-1 and R were significantly increased insubjects with AD compared to control subject serum exosomal levels.Further, neuron-derived serum exosomal P-S312-IRS-1, P-panY-IRS-1 and Rwere significantly increased in subjects as early as 10 years beforeclinical diagnosis of AD (see Table 8).

TABLE 8 Insulin IRS-1 P-S312-IRS-1 P-panY-IRS-1 Resistance Group (pg/ml)(U/ml) (AU) Index (R) AP (n = 22) 5.62 ± 130.5 9.22 ± 1.07* 0.119 ±0.016* 92.0 ± 1.07* AD (n = 22) N/A 9.70 ± 0.95* 0.113 ± 0.011*  101 ±19.8* Control N/A 2.93 ± 0.13  0.205 ± 0.006  14.7 ± 0.89  (n = 22) Mean± SEM; *p < 0.0001 compared to Control by paired t test.

These results showed that neuron-derived serum exosomal levels ofP-S312-IRS-1, P-panY-IRS-1 and R are increased in subjects withAlzheimer's disease and are useful for identifying subjects withAlzheimer's disease. These results also showed that neuron-derived serumexosomal levels of P-S312-IRS-1, P-panY-IRS-1 and R are increased insubjects as early as 10 years before clinical diagnosis of Alzheimer'sdisease. These results further showed that assays and methods of thepresent invention are useful for identifying a subject at risk of aneurodegenerative disorder (e.g., Alzheimer's disease). Additionally,these results showed that the assays and methods of the presentinvention may be useful for early detection and determining theprogression of Alzheimer's disease. These results further indicated thatmethods and compositions of the present invention are useful fordiagnosing Alzheimer's disease and other neurodegenerative disorders.

Example 11 Neuron-Derived Plasma Exosomal Protein Levels in HumanSubjects with a Neurodegenerative Disorder

Exosomal levels of cathepsin D (CTSD), type 1 lysosome-associatedmembrane protein (LAMP1), ubiquitinylated proteins (UBP) and heat-shockprotein 70 (HSP70) levels were assayed in human subjects as follows.Thirty milliliters of venous blood were collected from 26 subjects withAlzheimer's disease (AD), 16 subjects with frontotemporal dementia(FTD), and matched control subjects (Alzheimer's Controls—AC and

Frontotemporal Dementia Controls—FTC). Blood samples were incubated for10 min at room temperature and centrifuged for 15 min at 1500 g. Plasmawas aspirated and stored in aliquots at −80° C.

One-half ml of plasma was incubated with 0.15 ml of thromboplastin-D(Fisher Scientific, Inc., Hanover Park, Ill.) at room temperature for 1hour, followed by addition of 0.35 ml of calcium- and magnesium-freeDulbecco's balanced salt solution (DBS⁻²) with three-times the suggestedfinal concentrations of protease inhibitor cocktail (Roche AppliedSciences, Inc., Indianapolis, Ind.) and phosphatase inhibitor cocktail(Pierce Halt, Thermo Scientific, Inc., Rockford, Ill.). Aftercentrifugation at 1,500 g for 20 min, supernates were mixed with 252 μlof ExoQuick exosome precipitation solution (EXOQ; System Biosciences,Inc., Mountainview, Calif.), and incubated for 1 hour at 4° C. Resultantexosome suspensions were centrifuged at 1,500 g for 30 min at 4° C. andeach pellet was re-suspended in 250 μl of distilled water with inhibitorcocktails by vortex-mixing after a −80° C. freeze-thaw cycle beforeimmunochemical enrichment of exosomes from neural sources.

Each sample was incubated for 1 hour at 4° C. with 1 μg of mouseanti-human CD171 (L1CAM neural adhesion protein) biotinylated antibody(clone 5G3, eBioscience, San Diego, Calif.) in 50 μL of 3% BSA (1:3.33dilution of Blocker BSA 10% solution in PBS, Thermo Scientific, Inc.)with 50 μL of 3% BSA and incubation for 30 min at 4° C. Aftercentrifugation at 200 g for 10 min at 4° C. and removal of thesupernate, each pellet was resuspended in 0.5 ml of M-PER mammalianprotein extraction reagent (Thermo Scientific, Inc.), that had beenadjusted to pH 8.0 with 1 M Tris-HCl (pH 8.6) and contained thecocktails of protease and phosphatase inhibitors. These suspensions wereincubated at 37° C. for 20 min and vortex-mixed for 15 sec beforestorage at −80° C. until use in ELISAs.

Exosome proteins were quantified by ELISA kits for total ubiquitin(FIVEphoton Biochemicals, San Diego, Calif.), type 1 lysosome-associatedmembrane protein (LAMP1) (USBiological Life Sciences, Salem, Mass.),heat-shock protein 70 (HSP70) (Enzo Life Sciences, Farmingdale, N.Y.),cathepsin D (EMD Milipore Corp., Billerica, Mass.) and tetraspanningexosome marker human CD81 (Hölzel Diagnostika-Cusabio, Cologne, Germany)with verification of the CD81 antigen standard curve using humanpurified recombinant CD81 antigen (Origene Technologies, Inc.,Rockville, Md.), according to suppliers' directions. The mean value forall determinations of CD81 in each assay group was set at 1.00 and therelative values for each sample used to normalize their recovery.

The statistical significance of differences between group means forcross-sectional patient groups and between each patient group and theirrespective matched control groups was determined with an unpaired t testincluding a Bonferroni correction in the interpretation (GraphPad Prism6, La Jolla, Calif.). Separate discriminant classifier analyses wereconducted to define the best simple linear models for comparing group ADwith its control group (AC) and FTD with its control group (FTC). Thediscriminant analyses were performed step-wise with the Wilks' Lambdamethod. In each step, only variables with a minimum partial F of 3.84 toenter and 2.71 to remove were retained. Prior probabilities wereconsidered equal for all groups. Fisher Function Coefficients and withingroup covariances also were computed. Receiver operating characteristics(ROC) analyses were conducted under the non-parametric distributionassumption for Standard Error of Area to determine the performance ofclassifier models. For longitudinal analyses, the significance ofdifferences between serial values for AD patients taken before and afteronset of aMCI or dementia was calculated with a paired t test(GraphPad).

As shown in Table 9 below, neuron-derived serum exosomal CTSD, LAMP1,and UBP protein levels were significantly increased and HSP70 proteinlevels were significantly decreased in subjects with AD compared totheir respective control subject serum exosomal levels. Neuron-derivedserum exosomal CTSD protein levels were significantly increased andHSP70 protein levels were significantly decreased in subjects with FTDcompared to their respective control subject serum exosomal levels (seeTable 9).

TABLE 9 Group CTSD (ng/ml) LAMP1 (pg/ml) UBP (pg/ml) HSP70 (pg/ml) AD (n= 26) 17.7 ± 0.80*  1,808 ± 204*  477 ± 25.4*  246 ± 18.0* AC (n = 26)8.35 ± 0.27   946 ± 119 225 ± 10.1 394 ± 15.2 FTD (n = 16) 12.8 ± 0.75*1,071 ± 62.7 255 ± 11.5  165 ± 4.39* FTC (n = 16) 6.23 ± 0.15  1,147 ±88.9 228 ± 8.53 429 ± 15.6 Mean ± SEM; *p < 0.0005 compared to Controlby paired t test.

Step-wise discriminant analysis of AD and AC data resulted in a modelthat incorporated CTSD, then UBP and finally HSP-70, but not LAMP-1. ROCcurves of AD vs. AC showed an area under the curve (AUC) of 1.0 for bothCTSD and composite scores from the final model with correctclassification of 100% of AD patients. Similar ROC analyses correctlyclassified 100% of FTD vs. FTC controls and 95.8% of AD vs. FTD.

These results showed that neuron-derived serum exosomal levels of CTSD,LAMP1, and UBP protein levels are increased and HSP70 protein levels aredecreased in subjects with AD and are useful for identifying subjectswith AD. These results also demonstrated that neuron-derived serumexosomal levels of CTSD are increased and HSP70 protein levels aredecreased in subjects with FTD and are useful for identifying subjectswith FTD. These results further indicated that methods and compositionsof the present invention are useful for diagnosing Alzheimer's disease,frontotemporal dementia, and other neurodegenerative disorders.

Example 12 Neuron-Derived Serum Exosomal Protein Levels PredictDevelopment of Alzheimer's Disease in Human Subjects

Exosomal levels of cathepsin D (CTSD), type 1 lysosome-associatedmembrane protein (LAMP1), ubiquitinylated proteins (UBP) and heat-shockprotein 70 (HSP70) levels were assayed in human subjects as follows. Tenmilliliters of venous blood were collected from subjects (n=20) at twotime-points: the first at one to ten years before the subjects'diagnosis of Alzheimer's disease (Alzheimer's preclinical, AP) and thesecond at the time of initial diagnosis of Alzheimer's disease (AD).Venous blood samples were also collected from control subjects (n=20).Blood samples were processed and exosomes were isolated as described inExample 11 above.

As shown in Table 10 below, neuron-derived serum exosomal serum exosomalCTSD, LAMP-1, and UBP were significantly increased and HSP70 proteinlevels were significantly decreased in subjects with AD compared tocontrol subject serum exosomal levels. Further, neuron-derived serumexosomal CTSD, LAMP-1, and UBP were increased and HSP70 protein levelswere decreased in subjects as early as 10 years before clinicaldiagnosis of AD (see Table 10).

TABLE 10 Group CTSD (ng/ml) LAMP1 (pg/ml) UBP (pg/ml) HSP70 (pg/ml) AP18.4 ± 0.68* 2,638 ± 354* 364 ± 13.9* 244 ± 16.4* AD 19.0 ± 0.70* 2,080± 257* 347 ± 13.9* 250 ± 11.8* Control 8.50 ± 0.36  1,035 ± 119  206 ±7.46  392 ± 14.2  Mean ± SEM; *p < 0.0003 compared to Control by pairedt test.

These results showed that neuron-derived serum exosomal protein levelsof CTSD, LAMP-1, and UBP are increased and HSP70 are decreased insubjects with Alzheimer's disease and are useful for identifyingsubjects with Alzheimer's disease. These results also showed thatneuron-derived serum exosomal levels of CTSD, LAMP-1, and UBP areincreased and HSP70 are decreased in subjects as early as 10 yearsbefore clinical diagnosis of Alzheimer's disease. These results furthershowed that assays and methods of the present invention are useful foridentifying a subject at risk of a neurodegenerative disorder (e.g.,Alzheimer's disease). Additionally, these results showed that the assaysand methods of the present invention may be useful for early detectionand determining the progression of Alzheimer's disease. These resultsfurther indicated that methods and compositions of the present inventionare useful for diagnosing Alzheimer's disease and otherneurodegenerative disorders.

Example 13 Neuron-Derived Plasma Exosomal Protein Levels in HumanSubjects with a Neurodegenerative Disorder

Exosomal levels of neuron specific enolase (NSE) and neurofilament lightchain (NFL) levels were assayed in human subjects as follows. Thirtymilliliters of venous blood were collected from 20 subjects withAlzheimer's disease (AD) and matched control subjects (Alzheimer'sControls—AC). Blood samples were incubated for 10 min at roomtemperature and centrifuged for 15 min at 1500 g. Plasma was aspiratedand stored in aliquots at −80° C.

One-half ml of plasma was incubated with 0.15 ml of thromboplastin-D(Fisher Scientific, Inc., Hanover Park, Ill.) at room temperature for 1hour, followed by addition of 0.35 ml of calcium- and magnesium-freeDulbecco's balanced salt solution (DBS⁻²) with three-times the suggestedfinal concentrations of protease inhibitor cocktail (Roche AppliedSciences, Inc., Indianapolis, Ind.) and phosphatase inhibitor cocktail(Pierce Halt, Thermo Scientific, Inc., Rockford, Ill.). Aftercentrifugation at 1,500 g for 20 min, supernates were mixed with 252 μlof ExoQuick exosome precipitation solution (EXOQ; System Biosciences,Inc., Mountainview, Calif.), and incubated for 1 hour at 4° C. Resultantexosome suspensions were centrifuged at 1,500 g for 30 min at 4° C. andeach pellet was re-suspended in 250 μl of distilled water with inhibitorcocktails by vortex-mixing after a −80° C. freeze-thaw cycle beforeimmunochemical enrichment of exosomes from neural sources.

Each sample was incubated for 1 hour at 4° C. with 1 μg of mouseanti-human CD171 (L1CAM neural adhesion protein) biotinylated antibody(clone 5G3, eBioscience, San Diego, Calif.) in 50 μL of 3% BSA (1:3.33dilution of Blocker BSA 10% solution in PBS, Thermo Scientific, Inc.)with 50 μL of 3% BSA and incubation for 30 min at 4° C. Aftercentrifugation at 200 g for 10 min at 4° C. and removal of thesupernate, each pellet was resuspended in 0.5 ml of M-PER mammalianprotein extraction reagent (Thermo Scientific, Inc.), that had beenadjusted to pH 8.0 with 1 M Tris-HCl (pH 8.6) and contained thecocktails of protease and phosphatase inhibitors. These suspensions wereincubated at 37° C. for 20 min and vortex-mixed for 15 sec beforestorage at −80° C. until use in ELISAs.

Exosome proteins were quantified by ELISA kits for total neuron-specificenolase (R&D Systems, St. Paul, Minn.), neurofilament light chainprotein (NFL) (American Research Products, Waltham, Mass.), andtetraspanning exosome marker human CD81 (Hölzel Diagnostika-Cusabio,Cologne, Germany) with verification of the CD81 antigen standard curveusing human purified recombinant CD81 antigen (Origene Technologies,Inc., Rockville, Md.), according to suppliers' directions. The meanvalue for all determinations of CD81 in each assay group was set at 1.00and the relative values for each sample used to normalize theirrecovery.

The statistical significance of differences between group means forcross-sectional patient groups and between each patient group and theirrespective matched control groups was determined with an unpaired t testincluding a Bonferroni correction in the interpretation (GraphPad Prism6, La Jolla, Calif.). Separate discriminant classifier analyses wereconducted to define the best simple linear models for comparing group ADwith its control group (AC). The discriminant analyses were performedstep-wise with the Wilks' Lambda method. In each step, only variableswith a minimum partial F of 3.84 to enter and 2.71 to remove wereretained. Prior probabilities were considered equal for all groups.Fisher Function Coefficients and within group covariances also werecomputed. Receiver operating characteristics (ROC) analyses wereconducted under the non-parametric distribution assumption for StandardError of Area to determine the performance of classifier models.

As shown in Table 11 below, neuron-derived serum exosomal NSE and NFLprotein levels were significantly increased in subjects with AD comparedto their respective control subject serum exosomal levels.

TABLE 11 Group NSE (pg/ml) NFL (pg/ml) AD (n = 20) 6,968 ± 185* 1,296 ±62* AC (n = 20) 3,053 ± 134   317 ± 31 Mean ± SEM; *p < 0.0001 comparedto Control by paired t test.

These results showed that neuron-derived serum exosomal levels of NSEand NFL protein levels are increased in subjects with AD and are usefulfor identifying subjects with AD. These results further indicated thatmethods and compositions of the present invention are useful fordiagnosing Alzheimer's disease and other neurodegenerative disorders.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated by reference hereinin their entirety.

What is claimed is:
 1. A method of analyzing a sample from a subjectcomprising the steps of: (i) obtaining a biological sample comprisingvesicles from the subject, (ii) measuring the level of one or morebiomarkers in the biological sample, and (iii) comparing the level ofthe one or more biomarkers in the biological sample to a control levelof the one or more biomarkers in a control biological sample, wherein atleast one of the one or more biomarkers is selected from the groupconsisting of Tau, phosphorylated Tau, Aß1-42, TDP-43, α-synuclein,SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP,HSP70, NSE, NFL, CD9, CD63, CD81, and CD171.
 2. The method of claim 1,wherein the vesicles are selected from the group consisting of exosomes,microparticles, microvesicles, nanosomes, extracellular vesicles, andectosomes.
 3. The method of claim 2, wherein the exosomes are selectedfrom the group consisting of neuron-derived exosomes, astrocyte-derivedexosomes, oliogodendrocyte-derived exosomes, and microglia-derivedexosomes.
 4. The method of claim 1, further comprising isolatingvesicles from the biological sample.
 5. The method of claim 1, whereinthe level of one or more biomarkers is the protein, phosphorylatedprotein, mRNA, or miRNA level of the one or more biomarker.
 6. Themethod of claim 1, wherein the phosphorylated tau and/or phosphorylatedIRS-1 is phosphorylated on one or more serine, threonine, or tyrosineresidues.
 7. The method of claim 1, wherein the phosphorylated tauand/or phosphorylated IRS-1 is phosphorylated at one or more residuesselected from the group consisting of Thr-153, Thr-181, Thr-205,Thr-231, Ser-199, Ser-202, Ser-214, Ser-235, Ser-262, Ser-356, Ser-396,Ser-422, Tyr18, Tyr29, Tyr197, Tyr310, Tyr 394, P-S312-IRS-1 andP-panY-IRS-1.
 8. The method of claim 1, wherein the subject has beendiagnosed or suspected of having a neurodegenerative disorder.
 9. Themethod of claim 8, wherein the neurodegenerative disorder is selectedfrom the group consisting of: Alzheimer's disease (AD), vascular diseasedementia, frontotemporal dementia (FTD), corticobasal degeneration(CBD), progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease.
 10. The method of claim 1, wherein the biologicalsample is selected from the group consisting of whole blood, serum,plasma, urine, interstitial fluid, peritoneal fluid, cervical swab,tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.11. The method of claim 4, wherein the isolating vesicles from abiological sample comprises: contacting the biological sample with anagent under conditions wherein a vesicle present in said biologicalsample binds to said agent to form a vesicle-agent complex; andisolating said vesicle from said vesicle-agent complex to obtain asample containing said vesicle, wherein the purity of vesicles presentin said sample is greater than the purity of vesicles present in saidbiological sample.
 12. A set of biomarkers for assessingneurodegenerative disorder status of a subject comprising one or morebiomarkers, wherein the levels of the biomarkers in the set are assayed;and wherein the biomarker level determines the neurodegenerativedisorder status of the subject with at least 40% specificity, wherein atleast one or more of the set of biomarkers are selected from the groupconsisting of Tau, phosphorylated Tau, Aß1-42, TDP-43, α-synuclein,SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP,HSP70, NSE, NFL, CD9, CD63, CD81, and CD171.
 13. The set of biomarkersof claim 12, wherein the biological sample is selected from the groupconsisting of whole blood, serum, plasma, urine, interstitial fluid,peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, braintissue, and cerebrospinal fluid.
 14. The set of biomarkers of claim 12,wherein the neurodegenerative disorder is selected from the groupconsisting of: Alzheimer's disease (AD), vascular disease dementia,frontotemporal dementia (FTD), corticobasal degeneration (CBD),progressive supranuclear palsy (PSP), Lewy body dementia,tangle-predominant senile dementia, Pick's disease (PiD), argyrophilicgrain disease, amyotrophic lateral sclerosis (ALS), other motor neurondiseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodigdisease, multiple sclerosis, traumatic brain injury (TBI), andParkinson's disease.
 15. The set of biomarkers of claim 12, furthercomprising assaying the levels of the biomarkers in vesicles from thesample.
 16. The set of biomarkers of claim 15, wherein the vesicles areselected from the group consisting of exosomes, microparticles,microvesicles, nanosomes, extracellular vesicles, and ectosomes.